Carbohydrate purification using ultrafiltration, reverse osmosis and nanofiltration

ABSTRACT

The invention provides methods for purifying carbohydrates, including oligosaccharides, nucleotide sugars, and related compounds, by use of ultrafiltration, nanofiltration and/or reverse osmosis. The carbohydrates are purified away from undesired contaminants such as compounds present in reaction mixtures following enzymatic synthesis or degradation of oligosaccharides.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This is a continuation-in-part of United States ProvisionalApplication No. 60/028,226, filed Oct. 10, 1996, the disclosure of whichis incorporated by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to the synthesis ofoligosaccharides. In particular, it relates to improved methods forpurifying oligosaccharides using ultrafiltration, nanofiltration and/orreverse osmosis.

BACKGROUND OF THE INVENTION

[0003] Increased understanding of the role of carbohydrates asrecognition elements on the surface of cells has led to increasedinterest in the production of carbohydrate molecules of definedstructure. For instance, compounds comprising the oligosaccharidemoiety, sialyl lactose, have been of interest as neutralizers forenterotoxins from bacteria such as Vibrio cholerae, Escherichia coli,and Salmonella (see, e.g., U.S. Pat. No. 5,330,975). Sialyl lactose hasalso been investigated for the treatment of arthritis and relatedautoimmune diseases. In particular, sialyl lactose is thought to inhibitor disrupt the degree of occupancy of the Fc carbohydrate binding siteon IgG, and thus prevent the formation of immune complexes (see, U.S.Pat. No. 5,164,374). Recently, sialyl-α(2,3)galactosides, sialyl lactoseand sialyl lactosamine have been proposed for the treatment of ulcers,and Phase I clinical trials have begun for the use of the formercompound in this capacity. See, Balkonen et al., FEMS Immunology andMedical Microbiology 7:29 (1993) and BioWorld Today, p.5, Apr. 4, 1995.As another example, compounds comprising the sialyl Lewis ligands,sialyl Lewis^(x) and sialyl Lewis^(a) are present in leukocyte andnon-leukocyte cell lines that bind to receptors such as the ELAM-1 andGMP 140 receptors. Polley et al., Proc. Natl. Acad. Sci., USA, 88:6224(1991) and Phillips et al., Science, 250:1130 (1990), see, also, U.S.Ser. No. 08/063,181.

[0004] Because of interest in making desired carbohydrate structures,glycosyltransferases and their role in enzyme-catalyzed synthesis ofcarbohydrates are presently being extensively studied. The use ofglycosyltransferases for enzymatic synthesis of carbohydrate offersadvantages over chemical methods due to the virtually completestereoselectivity and linkage specificity offered by the enzymes (Ito etal., Pure Appl. Chem., 65:753 (1993) U.S. Pat. Nos. 5,352,670, and5,374,541). Consequently, glycosyltransferases are increasingly used asenzymatic catalysts in synthesis of a number of carbohydrates used fortherapeutic and other purposes.

[0005] Carbohydrate compounds produced by enzymatic synthesis or byother methods are often obtained in the form of complex mixtures thatinclude not only the desired compound but also contaminants such asunreacted sugars, salts, pyruvate, phosphate, PEP, nucleosides,nucleotides, and proteins, among others. The presence of thesecontaminants is undesirable for many applications for which thecarbohydrate compounds are useful. Previously used methods for purifyingoligosaccharides, such as chromatography, i.e., ion exchange and sizeexclusion chromatography, have several disadvantages. For example,chromatographic purification methods are not amenable to large-scalepurifications, thus precluding their use for commercial production ofsaccharides. Moreover, chromatographic purification methods areexpensive. Therefore, a need exists for purification methods that arefaster, more efficient, and less expensive than previously used methods.The present invention fulfills this and other needs.

BACKGROUND ART

[0006] A method for using a combination of membranes to removeundesirable impurities from a sugar-containing solution, especiallymolasses-forming ions which inhibit sugar crystallization is describedin U.S. Pat. No. 5,454,952. The method, which involves ultrafiltrationfollowed by nanofiltration, is described as being useful for improvingthe recovery of crystalline sugar from sugar cane or sugar beetsolutions.

[0007] U.S. Pat. No. 5,403,604 describes the removal of fruit juicesugars from fruit juice by nanofiltration to obtain a retentate having ahigh level of sugars and a permeate having a lower level of sugars.

[0008] U.S. Pat. No. 5,254,174 describes the use of chromatographyand/or nanofiltration to purify inulide compounds of formula GF. (whereG is glucose and F is fructose) by removing salts and glucose, fructose,and sucrose from a juice or syrup containing the inulide compounds.

[0009] U.S. Pat. No. 4,956,458 describes the use of reverse osmosis toremove from polydextrose, which is a randomly cross-linked glucanpolymer produced through the acid-catalyzed condensation of glucose,most of the off-flavor constituents such as anhydroglucose andfuraldehyde derivatives polydextrose.

[0010] U.S. Pat. No. 4,806,244 describes the use of a combined membraneand sorption system in which sulfate is removed from water bynanofiltration, after which the nitrate, which passed through themembrane, was removed from the permeate by absorption to an ion exchangeresin.

SUMMARY OF THE INVENTION

[0011] The present invention provides methods of purifying acarbohydrate compound from a feed solution containing a contaminant. Themethods involve contacting the feed solution with a nanofiltration orreverse osmosis membrane under conditions such that the membrane retainsthe desired carbohydrate compound while a majority of the contaminantpasses through the membrane. The invention provides methods forpurifying carbohydrate compounds such as sialyl lactosides, sialic acid,lacto-N-neotetraose (LNnT) and GlcNAcβ1,3Galβ1,4Glc (LNT-2),NeuAcα(2→3)Galβ(1→4)(Fucα1→3)Glc(R¹)β1-OR², wherein R¹ is OH or NAc; R²is a hydrogen, an alkoxy, a saccharide, an oligosaccharide or an aglycongroup having at least one carbon atom; andGalα(1→3)Galβ(1→4)Glc(R¹)β-O—R³, wherein R¹ is OH or NAc; R³ is—(CH₂)_(n)—COX, with X═OH, OR⁴, —NHNH₂, R⁴ being a hydrogen, asaccharide, an oligosaccharide or an aglycon group having at least onecarbon atom, and n=an integer from 2 to 18.

[0012] Also provided are methods for purifying carbohydrate compoundshaving a formula NeuAcα(2→3)Galβ(1→4)GlcN(R¹)β-OR²,NeuAcα(2→3)Galβ(1→4)GlcN(R¹)β(1→3)Galβ-OR², NeuAcα(2→3)Galβ(1→4)(Fucα1→3)GlcN(R¹)β-OR², or NeuAcα(2→3)Galβ(1→4)(Fucα1→3)GlcN(R¹)β(1→3)Galβ-OR², wherein R¹ is alkyl or acyl from 1-18carbons, 5,6,7,8-tetrahydro-2-naphthamido; benzamido; 2-naphthamido;4-aminobenzamido; or 4-nitrobenzamido, and R² is a hydrogen, asaccharide, an oligosaccharide or an aglycon group having at least onecarbon atom.

[0013] In another embodiment, the invention provides methods ofpurifying a carbohydrate compound from a feed solution comprising areaction mixture used to synthesize the carbohydrate compound. Thesynthesis can be enzymatic or chemical, or a combination thereof. Themethods involve removing any proteins present in the feed solution bycontacting the feed solution with an ultrafiltration membrane so thatproteins are retained the membrane while the carbohydrate compoundpasses through the membrane as a permeate. The permeate from theultrafiltration step is then contacted with a nanofiltration or reverseosmosis membrane under conditions such that the nanofiltration orreverse osmosis membrane retains the carbohydrate compound while amajority of an undesired contaminant passes through the membrane.

[0014] Another embodiment of the invention provides methods forpurifying nucleotides, nucleosides, and nucleotide sugars by contactinga feed solution containing the nucleotide or related compound with ananofiltration or reverse osmosis membrane under conditions such thatthe membrane retains the nucleotide or related compound while a majorityof the contaminant passes through the membrane.

[0015] The present invention also provides methods for removing one ormore contaminants from a solution that contains a carbohydrate ofinterest. The methods involve contacting the solution with a first sideof a semipermeable membrane having rejection coefficients so as toretain the carbohydrate while allowing the contaminant to pass throughthe membrane. The membrane is selected from the group consisting of anultrafiltration membrane, a nanofiltration membrane, and a reverseosmosis membrane, depending on the size and charge of the carbohydrateof interest relative to those of the contaminants. The membraneseparates a feed solution containing a carbohydrate into a retentateportion and a permeate portion. If the rejection coefficient of themembrane is greater for the carbohydrate than for the contaminant, theretentate portion will have a lower concentration of the contaminantrelative to the contaminant concentration in the feed solution, andgenerally also a higher ratio of the carbohydrate to the undesiredcontaminant. Conversely, a membrane having a rejection coefficient forthe carbohydrate that is lesser than that for the contaminant willeffect a separation wherein the concentration of the contaminant islower in the permeate than in the feed solution, and the permeate willhave a higher ratio of carbohydrate to contaminant than the feedsolution. If desired, the fraction containing the carbohydrate can berecycled through the membrane system for further purification.

[0016] Examples of contaminants that can be removed from solutionscontaining the compound of interest using the methods of the inventioninclude, but are not limited to, unreacted sugars, inorganic ions,pyruvate, phosphate, phosphoenolpyruvate, and proteins.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] Definitions

[0018] The following abbreviations are used herein:

[0019] Ara=arabinosyl;

[0020] Fru=fructosyl;

[0021] Fuc=fucosyl;

[0022] Gal=galactosyl;

[0023] GalNAc=N-acetylgalacto;

[0024] Glc=glucosyl;

[0025] GlcNAc=N-acetylgluco;

[0026] Man=mannosyl; and

[0027] NeuAc=sialyl (N-acetylneuraminyl).

[0028] The term “carbohydrate” encompasses chemical compounds having thegeneral formula (CH₂O)_(n), and includes monosaccharides, disaccharides,oligosaccharides, and polysaccharides. The term “oligo,” as used herein,refers to a polymeric molecule consisting of 2 to approximately 10residues, for example, amino acids (oligopeptide), monosaccharides(oligosaccharide), and nucleic acids (oligonucleotide). The term “poly”refers to a polymeric molecule comprising greater than about 10residues.

[0029] Oligosaccharides are considered to have a reducing end and anon-reducing end, whether or not the saccharide at the reducing end isin fact a reducing sugar. In accordance with accepted nomenclature,oligosaccharides are depicted herein with the non-reducing end on theleft and the reducing end on the right.

[0030] All oligosaccharides described herein are described with the nameor abbreviation for the non-reducing saccharide (e.g., Gal), followed bythe configuration of the glycosidic bond (α or β), the ring bond, thering position of the reducing saccharide involved in the bond, and thenthe name or abbreviation of the reducing saccharide (e.g., GlcNAc). Thelinkage between two sugars may be expressed, for example, as 2,3, 2→or(2,3).

[0031] A compound is “substantially purified” from an undesiredcomponent in a solution if the concentration of the undesired componentafter purification is no greater than about 40% of the concentration ofthe component prior to purification. Preferably, the post-purificationconcentration of the undesired component will be less than about 20% byweight, and more preferably less than about 10%, of the pre-purificationconcentration.

[0032] The term “pharmaceutically pure,” as used herein, refers to acompound that is sufficiently purified from undesired contaminants thatthe compound is suitable for administration as a pharmaceutical agent.Preferably, the compound is purified such that the undesired contaminantis present after purification in an amount that is about 5% by weight orless of the pre-purification concentration of the contaminant in thefeed solution. More preferably, the post-purification concentration ofthe contaminant is about 1% or less of the pre-purification contaminantconcentration, and most preferably about 0.5% or less of thepre-purification concentration of contaminant.

[0033] A “feed solution” refers to any solution that contains a compoundto be purified. For example, a reaction mixture used to synthesize anoligosaccharide can be used as a feed solution from which the desiredreaction product is purified using the methods of the invention.

[0034] Embodiments of the Invention

[0035] The present invention provides methods for rapidly andefficiently purifying specific carbohydrate and oligosaccharidestructures to a high degree of purity using semipermeable membranes suchas reverse osmosis and/or nanofiltration membranes. The methods areparticularly useful for separating desired oligosaccharide compoundsfrom reactants and other contaminants that remain in a reaction mixtureafter synthesis or breakdown of the oligosaccharides. For example, theinvention provides methods for separating oligosaccharides from enzymesand/or other components of reaction mixtures used for enzymaticsynthesis or enzymatic degradation of oligosaccharides, nucleotidesugars, glycolipids, liposaccharides, nucleotides, nucleosides, andother saccharide-containing compounds. Also provided are methods forremoving salts, sugars and other components from feed solutions usingultrafiltration, nanofiltration or reverse osmosis. Using thesetechniques, the saccharides (e.g., sialyl lactose, SLe^(x), and manyothers) can be produced at up to essentially 100% purity. Moreover, thepurification methods of the invention are more efficient, rapid, andamenable to large-scale purifications than previously known carbohydratepurification methods.

[0036] Often, a desired purification can be effected in a single step;additional purification steps such as crystallization and the like aregenerally not required. Accordingly, the invention provides single-stepmethods for purifying saccharide-containing compounds.

[0037] To purify saccharides according to the invention, a membrane isselected that is appropriate for separating the desired carbohydratefrom the undesired components of the solution from which thecarbohydrate is to be purified. The goal in selecting a membrane is tooptimize for a particular application the molecular weight cutoff(MWCO), membrane composition, permeability, and rejectioncharacteristics, that is, the membrane's total capacity to retainspecific molecules while allowing salts and other, generally smaller oropposite charged molecules, to pass through. The percent retention of acomponent i (R_(i)) is given by the formulaR_(i)=(1−C_(ip)/C_(ir))×100%, wherein C_(ip) is the concentration ofcomponent i in the permeate and C_(ir) is the concentration of componenti in the retentate, both expressed in weight percent. The percentretention of a component is also called the retention characteristic orthe membrane rejection coefficient.

[0038] For effective separation, a membrane is chosen-that has a highrejection ratio for the saccharide of interest relative to the rejectionratio for compounds from which separation is desired. If a membrane hasa high rejection ratio for a first compound relative to a secondcompound, the concentration of the first compound in the permeatesolution which passes through the membrane is decreased relative to thatof the second compound. Conversely, the concentration of the firstcompound increases relative to the concentration of the second compoundin the retentate. If a membrane does not reject a compound, theconcentration of the compound in both the permeate and the rejectportions will remain essentially the same as in the feed solution. It isalso possible for a membrane to have a negative rejection rate for acompound if the compound's concentration in the permeate becomes greaterthan the compound's concentration in the feed solution. A general reviewof membrane technology is found in “Membranes and Membrane SeparationProcesses,” in Ullmann's Encyclopedia of Industrial Chemistry (VCH,1990); see also, Noble and Stern, Membrane Separations Technology:Principles and Applications (Elsevier, 1995).

[0039] As a starting point, one will generally choose a membrane havinga molecular weight cut-off (MWCO, which is often related to membranepore size) that is expected to retain the desired compounds whileallowing an undesired compound present in the feed stream to passthrough the membrane. The desired MWCO is generally less than themolecular weight of the compound being purified, and is typicallygreater than the molecular weight of the undesired contaminant that isto be removed from the solution containing the compound being purified.For example, to purify a compound having a molecular weight of 200 Da,one would choose a membrane that has a MWCO of less than about 200 Da. Amembrane with a MWCO of 100 Da, for example, would also be a suitablecandidate. The membranes that find use in the present invention areclassified in part on the basis of their MWCO as ultrafiltration (UF)membranes, nanofiltration (NF) membranes, or reverse osmosis (RO)membranes, depending on the desired separation. For purposes of thisinvention, UF, NF, and RO membranes are classified as defined in thePure Water Handbook, Osmonics, Inc. (Minnetonka Minn.). RO membranestypically have a nominal MWCO of less than about 200 Da and reject mostions, NF membranes generally have a nominal MWCO of between about 150 Daand about 5 kDa, and UF membranes generally have a nominal MWCO ofbetween about 1 kDa and about 300 kDa (these MWCO ranges assume asaccharide-like molecule).

[0040] A second parameter that is considered in choosing an appropriatemembrane for a particular separation is the polymer type of themembrane. The membranes used in each zone are made of conventionalmembrane material whether inorganic, organic, or mixed inorganic andorganic. Typical inorganic materials include glasses, ceramics, cermets,metals and the like. Ceramic membranes, which are preferred for the UFzone, may be made, for example, as described in U.S. Pat. No. 4,692,354to Asaeda et al, U.S. Pat. No. 4,562,021 to Alary et al., and others.The organic materials which are preferred for the NF and RO zones, aretypically polymers, whether isotropic, or anisotropic with a thin layeror “skin” on either the bore side or the shell side of the fibers.Preferred materials for fibers are polyamides, polybenzamides,polysulfones (including sulfonated polysulfone and sulfonated polyethersulfone, among others), polystyrenes, including styrene-containingcopolymers such as acrylo-nitrile-styrene, butadiene-styrene andstyrene-vinylbenzylhalide copolymers, polycarbonates, cellulosicpolymers including cellulose acetate, polypropylene, poly(vinylchloride), poly(ethylene terephthalate), polyvinyl alcohol,fluorocarbons, and the like, such as those disclosed in U.S. Pat. Nos.4,230,463, 4,806,244, and 4,259,183. The NF and RO membranes oftenconsist of a porous support substrate in addition to the polymericdiscrimination layer.

[0041] Of particular importance in selecting a suitable membranecomposition is the membrane surface charge. Within the required MWCOrange, a membrane is selected that has a surface charge that isappropriate for the ionic charge of the carbohydrate and that of thecontaminants. While MWCO for a particular membrane is generallyinvariable, changing the pH of the feed solution can affect separationproperties of a membrane by altering the membrane surface charge. Forexample, a membrane that has a net negative surface charge at neutral pHcan be adjusted to have a net neutral charge simply by lowering the pHof the solution. An additional effect of adjusting solution pH is tomodulate the ionic charge on the contaminants and on the carbohydrate ofinterest. Therefore, by choosing a suitable membrane polymer type andpH, one can obtain a system in which both the contaminant and themembrane are neutral, facilitating pass-through of the contaminant. If,for instance, a contaminant is negatively charged at neutral pH, it isoften desirable to lower the pH of the feed solution to protonate thecontaminant. For example, removal of phosphate is facilitated bylowering the pH of the solution to about 3, which protonates thephosphate anion, allowing passage through a membrane. As shown inExample 5, a decrease in pH from 7.5 to 3.0 decreases the percentage ofGlcNAc passing through a polyamide membrane such as an Osmonics MX07 inthirty minutes from 70% to 28%, while increasing the pass percentage ofphosphate from 10% to 46% (see, Example 6, Table 5 for additionalexamples of the effect of pH change on passage rate of other compoundsthrough various nanofiltration membranes). For purification of ananionic carbohydrate, the pH will generally between about pH 1 and aboutpH 7. Conversely, if contaminant has a positive surface charge, the pHof the feed solution can be adjusted to between about pH 7 and about pH14. For example, increasing the pH of a solution containing acontaminant having an amino group (—NH₃ ⁺) will make the amino groupneutral, thus facilitating its passage through the membrane. Thus, oneaspect of the invention involves modulating a separation by adjustingthe pH of a solution in contact with the membrane; this can change theionic charge of a contaminant and can also affect the surface charge ofthe membrane, thus facilitating purification if the desiredcarbohydrate. Of course, the manufacturer's instructions must befollowed as to acceptable pH range for a particular membrane to avoiddamage to the membrane.

[0042] For some applications, a mixture is first subjected tonanofiltration or reverse osmosis at one pH, after which the retentatecontaining the saccharide of interest is adjusted to a different pH andsubjected to an additional round of membrane purification. For example,filtration of a reaction mixture used to synthesize sialyl lactosethrough an Osmonics MX07 membrane (a nanofiltration membrane having aMWCO of about 500 Da) at pH 3.0 will retain the sialyl lactose andremove most phosphate, pyruvate, salt and manganese from the solution,while also removing some of the GlcNAc, lactose, and sialic acid.Further recirculation through the MX07 membrane after adjusting the pHof the retentate to 7.4 will remove most of the remaining phosphate, allof the pyruvate, all of the lactose, some of the sialic acid, andsubstantial amounts of the remaining manganese.

[0043] If a saccharide is to be purified from a mixture that containsproteins, such as enzymes used to synthesize a desired oligosaccharideor nucleotide sugar, it is often desirable to remove the proteins as afirst step of the purification procedure. For a saccharide that issmaller than the proteins, this separation is accomplished by choosing amembrane that has an MWCO which is less than the molecular mass of theprotein or other macromolecule to be removed from the solution, but isgreater than the molecular mass of the oligosaccharide being purified(i.e., the rejection ratio in this case is higher for the protein thanfor the desired saccharide). Proteins and other macromolecules that havea molecular mass greater than the MWCO will thus be rejected by themembrane, while the saccharide will pass through the membrane.Conversely, if an oligosaccharide or nucleotide sugar is to be purifiedfrom proteins that are smaller than the oligosaccharide or nucleotidesugar, a membrane is used that has a MWCO that is larger than themolecular mass of the protein but smaller than that of theoligosaccharide or nucleotide sugar. Generally, separation of proteinsfrom carbohydrates will employ membranes that are commonly referred toas ultrafiltration (UF) membranes. UF membranes that are suitable foruse in the methods of the invention are available from severalcommercial manufacturers, including Millipore Corp. (Bedford, Mass.),Osmonics, Inc. (Minnetonka, Minn.), Filmtec (Minneapolis, Minn.), UOP,Desalination Systems, Advanced Membrane Technologies, and Nitto.

[0044] The invention also provides methods for removing salts and otherlow molecular weight components from a mixture containing a saccharideof interest by using a nanofiltration (NF) or a reverse osmosis (RO)membrane. Nanofiltration membranes are a class of membranes for whichseparation is based both on molecular weight and ionic charge. Thesemembranes typically fall between reverse osmosis and ultrafiltrationmembranes in terms of the size of species that will pass through themembrane. Nanofiltration membranes typically have micropores or openingsbetween chains in a swollen polymer network. Molecular weight cut-offsfor non-ionized molecules are typically in the range from 100-20,000Daltons. For ions of the same molecular weight, membrane rejections(retentions) will increase progressively for ionic charges of 0, 1, 2, 3etc. for a particular membrane because of increasing charge density(see, e.g., Eriksson, P., “Nanofiltration Extends the Range of MembraneFiltration,” Environmental Progress, 7: 58-59 (1988)). Nanofiltration isalso described in Chemical Engineering Progress, pp. 68-74 (March 1994),Rautenbach et al., Desalination 77: 73 (1990), and U.S. Pat. No.4,806,244). In a typical application, saccharides of interest will beretained by the nanofiltration membrane and contaminating salts andother undesired components will pass through. A nanofiltration membraneuseful in the methods of the invention will typically have a retentioncharacteristic for the saccharide of interest of from about 40% to about100%, preferably from about 70% to about 100%. The nanofilter membranesused in the invention can be any one of the conventional nanofiltermembranes, with polyamide membranes being particularly suitable. Severalcommercial manufacturers, including Millipore Corp. (Bedford, Mass.),Osmonics, Inc. (Minnetonka, Minn.), Filmtec, UOP, Advanced MembraneTechnologies, Desalination Systems, and Nitto, among others, distributenanofiltration membranes that are suitable for use in the methods of theinvention. For example, suitable membranes include the Osmonics MX07,YK, GH (G-10), GE (G-5), and HL membranes, among others.

[0045] Reverse osmosis (RO) membranes also allow a variety of aqueoussolutes to pass through them while retaining selected molecules.Generally, osmosis refers to a process whereby a pure liquid (usuallywater) passes through a semipermeable membrane into a solution (usuallysugar or salt and water) to dilute the solution and achieve osmoticequilibrium between the two liquids. In contrast, reverse osmosis is apressure driven membrane process wherein the application of externalpressure to the membrane system results in a reverse flux with the watermolecules passing from a saline or sugar solution compartment into thepure water compartment of the membrane system. A RO membrane, which issemipermeable and non-porous, requires an aqueous feed to be pumped toit at a pressure above the osmotic pressure of the substances dissolvedin the water. An RO membrane can effectively remove low molecular weightmolecules (<200 Daltons) and also ions from water. Preferably, thereverse osmosis membrane will have a retention characteristic for thesaccharide of interest of from about 40% to about 100%, preferably fromabout 70% to about 100%. Suitable RO membranes include, but are notlimited to, the Filmtec BW-30, Filmtec SW-30, Filmtec SW-30HR, UOP ROmembranes, Desal RO membranes, Osmonics RO membranes, Advanced MembraneTechnologies RO membranes, and the Nitto RO membranes, among others. Oneexample of a suitable RO membrane is Millipore Cat. No. CDRN500 60(Millipore Corp., Bedford Mass.).

[0046] The membranes used in the invention may be employed in any of theknown membrane constructions. For example, the membranes can be flat,plate and frame, tubular, spiral wound, hollow fiber, and the like. In apreferred embodiment, the membrane is spiral wound. The membranes can beemployed in any suitable configuration, including either a cross-flow ora depth configuration. In “cross-flow” filtration, which is preferredfor ultrafiltration, nanofiltration and reverse osmosis purificationsaccording to the invention, the “feed” or solution from which thecarbohydrate of interest is to be purified flows through membranechannels, either parallel or tangential to the membrane surface, and isseparated into a retentate (also called recycle or concentrate) streamand a permeate stream. To maintain an efficient membrane, the feedstream should flow, at a sufficiently high velocity, parallel to themembrane surface to create shear forces and/or turbulence to sweep awayaccumulating particles rejected by the membrane. Cross-flow filtrationthus entails the flow of three streams—feed, permeate and retentate. Incontrast, a “dead end” or “depth” filter has only two streams—feed andfiltrate (or permeate). The recycle or retentate stream, which retainsall the particles and large molecules rejected by the membrane, can beentirely recycled to the membrane module in which the recycle stream isgenerated, or can be partially removed from the system. When the methodsof the invention are used to purify saccharides from lower molecularweight components, for example, the desired saccharides are contained inthe retentate stream (or feed stream, for a depth filter), while thepermeate stream contains the removed contaminants.

[0047] The purification methods of the invention can be furtheroptimized by adjusting the pressure, flow rate, and temperature at whichthe filtration is carried out. UF, NF, and RO generally requireincreasing pressures above ambient to overcome the osmotic pressure ofthe solution being passed through the membrane. The membranemanufacturers'instructions as to maximum and recommended operatingpressures can be followed, with further optimization possible by makingincremental adjustments. For example, the recommended pressure for UFwill generally be between about 25 and about 100 psi, for NF betweenabout 50 psi and about 1500 psi, and for RO between about 100 and about1500 psi. Flow rates of both the concentrate (feed solution) and thepermeate can also be adjusted to optimize the desired purification.Again, the manufacturers' recommendations for a particular membraneserve as a starting point from which to begin the optimization processby making incremental adjustments. Typical flow rates for theconcentrate (P_(c)) will be between about 1 and about 15 gallons perminute (GPM), and more preferably between about 3 and about 7 GPM. Forthe permeate, flow rates P_(f)) of between about 0.05 GPM and about 10GPM are typical, with flow rates between about 0.2 and about 1 GPM beingpreferred. The temperature at which the purification is carried out canalso influence the efficiency and speed of the purification.Temperatures of between about 0 and about 100° C. are typical, withtemperatures between about 20 and 40° C. being preferred for mostapplications. Higher temperatures can, for some membranes, result in anincrease in membrane pore size, thus providing an additional parameterthat one can adjust to optimize a purification.

[0048] In a preferred embodiment, the filtration is performed in amembrane purification machine which provides a means for automatingcontrol of flow rate, pressure, temperature, and other parameters thatcan affect purification. For example, the Osmonics 213T membranepurification machine is suitable for use in the methods of theinvention, as are machines manufactured by other companies listed above.

[0049] The membranes can be readily cleaned either after use or afterthe permeability of the membrane diminishes. Cleaning can be effected ata slightly elevated temperature if so desired, by rinsing with water ora caustic solution. If the streams contain small amounts of enzyme,rinsing in the presence of small amounts of surfactant, for instanceULTRASIL•, might be useful. Also, one can use prefilters (100-200 μm) toprotect the more expensive nanofiltration membranes. Other cleaningagents can, if desired, be used. The choice of cleaning method willdepend on the membrane being cleaned, and the membrane manufacturer'sinstructions should be consulted. The cleaning can be accomplished witha forward flushing or a backward flushing.

[0050] The purification methods of the invention can be used alone or incombination with other methods for purifying carbohydrates. For example,an ion exchange resin can be used to remove particular ions from amixture containing a saccharide of interest, either before or afternanofiltration/reverse osmosis, or both before and after filtration. Ionexchange is particularly desirable if it is desired to remove ions suchas phosphate and nucleotides that remain after a first round ofnanofiltration or reverse osmosis. In the case of sialyl lactosesynthesis as discussed above, this can be accomplished, for example, byadding an anion exchange resin such as AG1X-8 (acetate form, BioRad;see, e.g, BioRad catalog for other ion exchange resins) to a retentatethat is at about pH 3.0 or lower until the phosphate concentration isreduced as desired. In this process, acetic acid is released, so one maywish to follow the ion exchange with an additional purification throughthe nanofiltration or reverse osmosis system. For example, one cancirculate the pH 3.0 or lower solution through an Osmonics MX07 orsimilar membrane until the conductivity of the permeate is low andstabilized. The pH of the solution can then be raised to 7.4 with NaOHand the solution recirculated through the same membrane to removeremaining sodium acetate and salt. Cations can be removed in a similarmanner; for example, to remove Mn²⁺, an acidic ion exchange resin can beused, such as AG50WX8 (H⁺) (BioRad).

[0051] The purification methods of the invention are particularly usefulfor purifying oligosaccharides that have been prepared using enzymaticsynthesis. Enzymatic synthesis using glycosyltransferases provides apowerful method for preparing oligosaccharides; for some applications itis desirable to purify the oligosaccharide from the enzymes and otherreactants in the enzymatic synthesis reaction mixture. Preferred methodsfor producing many oligosaccharides involve glycosyl transferase cycles,which produce at least one mole of inorganic pyrophosphate for each moleof product formed and are typically carried out in the presence of adivalent metal ion. Examples of glycosyltransferase cycles are thesialyltransferase cycles, which use one or more enzymes as well as otherreactants. See, e.g., U.S. Pat. No. 5,374,541 WO 9425615 A,PCT/US96/04790, and PCT/US96/04824. For example, a reaction used forsynthesis of sialylated oligosaccharides can contain asialyltransferase, a CMP-sialic acid synthetase, a sialic acid, anacceptor for the sialyltransferase, CTP, and a soluble divalent metalcation. An exemplary α(2,3)sialyltransferase referred to asa(2,3)sialtransferase (EC 2.4.99.6) transfers sialic acid to thenon-reducing terminal Gal of a Galβ1→3Glc disaccharide or glycoside.See, Van den Eijnden et al., J. Biol. Chem., 256:3159 (1981), Weinsteinet al., J. Biol. Chem., 257:13845 (1982) and Wen et al., J. Biol. Chem.,267:21011 (1992). Another exemplary α2,3-sialyltransferase (EC 2.4.99.4)transfers sialic acid to the non-reducing terminal Gal of thedisaccharide or glycoside. See, Rearick et al., J. Biol. Chem., 254:4444(1979) and Gillespie et al, J. Biol. Chem., 267:21004 (1992). Furtherexemplary enzymes include Gal-β-1,4-GlcNAc α-2,6 sialyltransferase (See,Kurosawa et al. Eur. J. Biochem. 219: 375-381 (1994)). The reactionmixture will also contain an acceptor for the sialyltransferase,preferably having a galactosyl unit. Suitable acceptors, include, forexample, Galβ1_(→)3GalNAc, lacto-N-tetraose, Galβ1_(→)3GlcNAc,Galβ1_(→)3Ara, Galβ1_(→)6GlcNAc, Galβ1_(→)4Glc (lactose),Galβ1_(→)4Glcβ1-OCH₂CH₃, Galβ1_(→)4Glcβ1-OCH₂CH₂CH₃,Galβ1_(→)4Glcβ1-OCH₂C₆H₅, Galβ1_(→)4GlcNAc, Galβ1-OCH₃, melibiose,raffinose, stachyose, and lacto-N-neotetraose (LNnT). The sialic acidpresent in the reaction mixture can include not only sialic acid itself(5-N-acetylneuraminic acid;5-N-acetylamino-3,5-dideoxy-D-glycero-D-galacto-2-nonulosonic acid;NeuAc, and sometimes also abbreviated AcNeu or NANA), but also9-substituted sialic acids such as a 9-O-C₁-C₆ acyl-NeuAc like9-O-lactyl-NeuAc or 9-O-acetyl-NeuAc, 9-deoxy-9-fluoro-NeuAc and9-azido-9-deoxy-NeuAc. The synthesis and use of these compounds in asialylation procedure is described in international application WO92/16640, published Oct. 1, 1992.

[0052] In preferred embodiments the reaction medium can further comprisea CMP-sialic acid recycling system comprising at least 2 moles ofphosphate donor per each mole of sialic acid, and catalytic amounts ofan adenine nucleotide, a kinase capable of transferring phosphate fromthe phosphate donor to nucleoside diphosphates, and a nucleosidemonophosphate kinase capable of transferring the terminal phosphate froma nucleoside triphosphate to CMP. For example, a suitable CMP-sialicacid regenerating system comprises cytidine monophosphate (CMP), anucleoside triphosphate (for example adenosine triphosphate (ATP), aphosphate donor (for example, phosphoenolpyruvate or acetyl phosphate),a kinase (for example, pyruvate kinase or acetate kinase) capable oftransferring phosphate from the phosphate donor to nucleosidediphosphates and a nucleoside monophosphate kinase (for example,myokinase) capable of transferring the terminal phosphate from anucleoside triphosphate to CMP. The previously discussedα(2,3)sialyltransferase and CMP-sialic acid synthetase can also beformally viewed as part of the CMP-sialic acid regenerating system. Forthose embodiments in which a CMP-sialic acid recycling system is notused, the reaction medium will preferably further comprise aphosphatase.

[0053] Pyruvate is a byproduct of the sialyltransferase cycle and can bemade use of in another reaction in which N-acetylmannosamine (ManNAc)and pyruvate are reacted in the presence of NeuAc aldolase (EC 4.1.3.3)to form sialic acid. Alternatively, advantage can be taken of theisomerization of GlcNAc to ManNAc, and the less expensive GlcNAc can beused as the starting material for sialic acid generation. Thus, thesialic acid can be replaced by ManNAc (or GlcNAc) and a catalytic amountof NeuAc aldolase. Although NeuAc aldolase also catalyzes the reversereaction (NeuAc to ManNAc and pyruvate), the produced NeuAc isirreversibly incorporated into the reaction cycle via CMP-NeuAccatalyzed by CMP-sialic acid synthetase. In addition, the startingmaterial, ManNAc, can also be made by the chemical conversion of GlcNAcusing methods known in the art (see, e.g., Simon et al., J. Am. Chem.Soc. 110:7159 (1988). The enzymatic synthesis of sialic acid and its9-substituted derivatives and the use of a resulting sialic acid in adifferent sialylating reaction scheme is disclosed in Internationalapplication WO 92/16640, published on Oct. 1, 1992, and incorporatedherein by reference.

[0054] When a galactosyltransferase is used for enzymatic synthesis ofan oligosaccharide, the reaction medium will preferably contain, inaddition to a galactosyltransferase, donor substrate, acceptor sugar anddivalent metal cation, a donor substrate recycling system comprising atleast 1 mole of glucose-1-phosphate per each mole of acceptor sugar, aphosphate donor, a kinase capable of transferring phosphate from thephosphate donor to nucleoside diphosphates, and a pyrophosphorylasecapable of forming UDP-glucose from UTP and glucose-1-phosphate andcatalytic amounts of UDP and a UDP-galactose-4-epimerase. Exemplarygalactosyltransferases include α(1,3) galactosyltransferase (E.C. No.2.4.1.151, see, e.g., Dabkowski et al., Transplant Proc. 25: 2921 (1993)and Yamamoto et al., Nature 345:229-233 (1990)) and β(1,4)galactosyltransferase (E.C. No. 2.4.1.38).

[0055] Oligosaccharides synthesized by other enzymatic methods can alsobe purified by the methods of the invention. For example, the methodsare useful for purifying oligosaccharides produced in non-cyclic orpartially cyclic reactions such as simple incubation of an activatedsaccharide and an appropriate acceptor molecule with aglycosyltransferase under conditions effective to transfer andcovalently bond the saccharide to the acceptor molecule.Glycosyltransferases, which include those described in, e.g., U.S. Pat.No. 5,180,674, and International Patent Publication Nos. WO 93/13198 andWO 95/02683, as well the glycosyltransferases encoded by the los locusof Neisseria (see, U.S. Pat. No. 5,545,553), can be bound to a cellsurface or unbound. Oligosaccharides that can be obtained using theseglycosyltransferases include, for example, Galα(1_(→)4)Galβ(1_(→)4)Glc,GlcNAcβ(1,3)Galβ(1,4)Glc, Galβ(1_(→)4)GlcNAcβ(1_(→)3)Galβ(1_(→)4) Glc,and GalNAcβ(1_(→)3)Galβ(1_(→)4)GlcNAcβ(1_(→)3) Galβ(1_(→)4)Glc, amongmany others.

[0056] Among the compounds that one can purify using the describedmethods are sialic acid and any sugar having a sialic acid moiety. Theseinclude the sialyl galactosides, including the sialyl lactosides, aswell as compounds having the formula:

NeuAcα(2_(→)3)Galβ(1_(→)4)GlcN(R′)β-OR

[0057] or

NeuAcα(2_(→)3)Galβ(1_(→)4)GlcN(R¹)β(1_(→)3)Galβ-OR

[0058] In these formulae, R′ is alkyl or acyl from 1-18 carbons,5,6,7,8-tetrahydro-2-naphthamido; benzamido; 2-naphthamido;4-aminobenzamido; or 4-nitrobenzamido. R is a hydrogen, a alkyl C₁-C₆, asaccharide, an oligosaccharide or an aglycon group having at least onecarbon atom. The term “aglycon group having at least one carbon atom”refers to a group —A—Z, in which A represents an alkylene group of from1 to 18 carbon atoms optionally substituted with halogen, thiol,hydroxy, oxygen, sulfur, amino, imino, or alkoxy; and Z is hydrogen,—OH, —SH, —NH₂, —NHR¹, —N(R¹)₂, —CO₂H, —CO₂R¹, —CONH₂, —CONHR¹,—CON(R¹)₂,—CONHNH₂, or —OR¹ wherein each R¹ is independently alkyl offrom 1 to 5 carbon atoms. In addition, R can be

[0059] where n,m,o=1-18; (CH₂)_(n)-R² (in which n=0-18), wherein R² is avariously substituted aromatic ring, preferably, a phenyl group, beingsubstituted with one or more alkoxy groups, preferably methoxy orO(CH₂)_(m)CH₃, (in which m=0-18), or a combination thereof. R can alsobe 3-(3,4,5-trimethoxyphenyl)propyl.

[0060] The present invention is also useful for purifying a variety ofcompounds that comprise selectin-binding carbohydrate moieties. Theseselectin-binding moieties have the general formula:

R¹ Galβ1,m(Fucα1,n)GlcNR⁰(R²)p—

[0061] in which R⁰ is (C₁-C₈ alkyl)carbonyl, (C₁-C₈ alkoxy)carbonyl, or(C₂-C₉ alkenyloxy)carbonyl, R¹ is an oligosaccharide or a group havingthe formula

[0062] R³ and R⁴ may be the same or different and may be H, C₁-C₈ alkyl,hydroxy-(C₁-C₈ alkyl), aryl-(C₁-C₈ alkyl), or (C₁-C₈ alkoxy)-(C₁-C₈alkyl), substituted or unsubstituted. R² may be H, C₁-C₈ alkyl,hydroxy-(C₁-C₈ alkyl), aryl-(C₁-C₈ alkyl), (C₁-C₈ alkyl)-aryl,alkylthio, α1,2Man, α1,6GalNAc, β1,3Galβ1,4Glc, α1,2Man-R⁸,α1,6GalNAc-R⁸, and β1,3Gal-R⁸. R⁸may be H, C₁-C₈ alkyl, C₁-C₈, alkoxy,hydroxy-(C₁-C₈ alkyl), aryl-(C₁-C₈ alkyl), (C₁-C₈ alkyl)-aryl, oralkylthio. In the formula, m and n are integers and may be either 3 or4; p may be zero or 1.

[0063] The substituted groups mentioned above may be substituted byhydroxy, hydroxy(C₁-C₄ alkyl), polyhydroxy(C₁-C₄ alkyl), alkanoamido, orhydroxyalknoamido substituents. Preferred substituents include hydroxy,polyhydroxy(C₃ alkyl), acetamido and hydroxyacetamido. A substitutedradical may have more than one substitution, which may be the same ordifferent.

[0064] For embodiments in which R¹ is an oligosaccharide, theoligosaccharide is preferably a trisaccharide. Preferred trisaccharidesinclude NeuAcα2,3Galβ1,4GlcNAcβ1,3 or NeuGcα2,3Galβ1,4GlcNAcβ1,3.

[0065] For embodiments in which R¹ is the group having the formula

[0066] R³ and R⁴ preferably form a single radical having the formula

—R⁵— or —(R⁶)_(q)—O—(R⁷)_(r)—

[0067] in which R⁵ is C₃-C₇ divalent alkyl, substituted orunsubstituted, R⁶ and R⁷ are the same or different and are C₁-C₆divalent alkyl, substituted or unsubstituted. In the formula, q and rare integers which may be the same or different and are either zeroor 1. The sum of q and r is always at least 1.

[0068] A more preferred structure for a single radical formed by R³ andR⁴ is one having the formula

—(R⁶)—O—

[0069] in which R⁶ is C₃-C₄ divalent alkyl, substituted orunsubstituted. For instance, R⁶ may have the formula —CH₂CH₂CH₂CH₂—,preferably substituted. The radical can be substituted with hydroxy,polyhydroxy(C₃ alkyl), and substituted or unsubstituted alkanoamidogroups, such as acetamido or hydroxyacetamido. The substituted structurewill typically form a monosaccharide, preferably a sialic acid such asNeuAc or NeuGc linked α2,3 to the Gal residue.

[0070] In the general formula, above, both m and n are integers and canbe either 3 or 4. Thus, in one set of structures Gal is linked β1,4 andFuc is linked α1,3 to GlcNAc. This formula includes the SLe^(x)tetrasaccharide. SLe^(x) has the formulaNeuAcα2,3Galβ1,4(Fucα1,3)GlcNAcβ1—. This structure is selectivelyrecognized by LECCAM-bearing cells. SLe^(x) compounds that can bepurified using the methods of the invention includeNeuAcα2,3Galβ1,4(Fucα1,3)GlcNAcβ1-Gal-OEt,NeuAcα2,3Galβ1,4(Fucα1,3)GlcNAcβ1,4Galβ1-OEt, and others that aredescribed in international application WO 91/19502. Other compounds thatone can purify using the methods include those described in US Pat. No.5,604,207 having the formula

[0071] Y is selected from the group consisting of C(O), SO₂, HNC(O),OC(O) and SC(O);

[0072] R¹ is selected from the group consisting of an aryl, asubstituted aryl and a phenyl C₁-C₃ alkylene group, wherein said arylsubstitutent is selected from the group consisting of a halo,trifuloromethyl, nitro, C₁-C₁₈ alkyl, C₁-C₁₈ alkoxy, amino, mono-C₁-C₁₈alkylamino, di-C₁-C₁₈ alkylamino, benzylamino, C₁-C₁₈ alkylbenzylamino,C₁-C₁₈ thioaklyl and C₁-C₁₈ alkyl carboxamido groups, or

[0073] R¹Y is allyloxycarbonyl or chloroacetyl;

[0074] R² is selected from the group consisting of monosaccharide(including β1,3Gal—OR, where R═H, alkyl, aryl or acyl), disaccharide,hydrogen, C₁-C₁₈, straight chain, branched chain or cyclic hydrocarbyl,C₁-C₆ alkyl, 3-(3,4,5-trimethoxyphenyl)propyl, C₁-C₅ alkyleneω-carboxylate, ω-trisubstituted silyl C₂-C₄ alkylene wherein saidω-trisubstituted silyl is a silyl group having three substituentsindependently selected from the group consisting of C₁-C₄ alkyl, phenyl,

[0075] or OR² together form a C₁-C₁₈ straight chain, branched chain orcyclic hydrocarbyl carbamate;

[0076] R³ is hydrogen or C₁-C₆ acyl;

[0077] R⁴ is hydrogen, C₁-C₆ alkyl or benzyl;

[0078] R⁵ is selected from the group consisting of hydrogen, benzyl,methoxybenzyl, dimethoxybenzyl and C₁-C₆ acyl;

[0079] R⁷ is methyl or hydroxymethyl; and

[0080] X is selected from the group consisting of C₁-C₆ acyloxy, C₂-C₆hydroxylacyloxy, hydroxy, halo and azido.

[0081] A related set of structures included in the general formula arethose in which Gal is linked β1,3 and Fuc is linked α1,4. For instance,the tetrasaccharide, NeuAcα2,3Galβ1,3(Fucα1,4)GlcNAcβ1-, termed hereSLe^(a), is recognized by selectin receptors. See, Berg et al., J. Biol.Chem., 266:14869-14872 (1991). In particular, Berg et al. showed thatcells transformed with E-selectin cDNA selectively boundneoglycoproteins comprising SLe^(a).

[0082] The methods of the invention are also useful for purifyingoligosaccharide compounds having the general formula Galα1,3Gal-,including Galα1,3Galβ1,4Glc(R)β-O—R¹, wherein R¹ is —(CH₂)_(n)—COX, withX═OH, OR², —NHNH₂, R═OH or NAc, and R² is a hydrogen, a saccharide, anoligosaccharide or an aglycon group having at least one carbon atom, andn=an integer from 2 to 18, more preferably from 2 to 10. For example,one can purify a compound having the formulaGalα1,3Galβ1,4GlcNAcβ-O—(CH₂)₅—COOH using procedures such as thosedescribed in Examples 7-8. Also among the compounds that can be purifiedaccording to the invention are lacto-N-neotetraose (LNnT),GlcNAcβ1,3Galβ1,4Glc (LNT-2), sialyl(α2,3)-lactose, andsialyl(α2,6)-lactose.

[0083] In the above descriptions, the terms are generally used accordingto their standard meanings. The term “alkyl” as used herein means abranched or unbranched, saturated or unsaturated, monovalent ordivalent, hydrocarbon radical having from 1 to 20 carbons, includinglower alkyls of 1-8 carbons such as methyl, ethyl, n-propyl, butyl,n-hexyl, and the like, cycloalkyls (3-7 carbons), cycloalkylmethyls (4-8carbons), and arylalkyls. The term “alkoxy” refers to alkyl radicalsattached to the remainder of the molecule by an oxygen, e.g., ethoxy,methoxy, or n-propoxy. The term “alkylthio” refers to alkyl radicalsattached to the remainder of the molecule by a sulfur. The term of“acyl” refers to a radical derived from an organic acid by the removalof the hydroxyl group. Examples include acetyl, propionyl, oleoyl,myristoyl.

[0084] The term “aryl” refers to a radical derived from an aromatichydrocarbon by the removal of one atom, e.g., phenyl from benzene. Thearomatic hydrocarbon may have more than one unsaturated carbon ring,e.g., naphthyl.

[0085] The term “alkoxy” refers to alkyl radicals attached to theremainder of the molecule by an oxygen, e.g., ethoxy, methoxy, orn-propoxy.

[0086] The term “alkylthio” refers to alkyl radicals attached to theremainder of the molecule by a sulfur.

[0087] An “alkanoamido” radical has the general formula —NH—CO—(C₁-C₆alkyl) and may or may not be substituted. If substituted, thesubstituent is typically hydroxyl. The term specifically includes twopreferred structures, acetamido, —NH—CO—CH₃, and hydroxyacetamido,—NH—CO—CH₂—OH.

[0088] The term “heterocyclic compounds” refers to ring compounds havingthree or more atoms in which at least one of the atoms is other thancarbon (e.g., N, O, S, Se, P, or As). Examples of such compounds includefurans (including the furanose form of pentoses, such as fucose), pyrans(including the pyranose form of hexoses, such as glucose and galactose)pyrimidines, purines, pyrazines and the like.

[0089] The methods of the invention are useful not only for purifyingcarbohydrates that that are newly synthesized, but also those that arethe products of degradation, e.g., enzymatic degradation. See, e.g.,Sinnott, M. L., Chem. Rev. 90: 1171-1202 (1990) for examples of enzymesthat catalyze degradation of oligosaccharides.

[0090] The invention also provides methods for purifying nucleotides,nucleotide sugars, and related compounds. For example, a nucleotidesugar such as GDP-fucose, GDP-mannose, CMP-NeuAc, UDP-glucose,UDP-galactose, UDP-N-acetylgalactosamine, and the like, can be purifiedby the methods described herein. The methods are also useful forpurifying nucleotides and nucleotides in various states ofphosphorylation (e.g., CMP, CDP, CTP, GMP, GDP, GTP, TMP, TDP, TTP, AMP,ADP, ATP, UMP, UDP, UTP), as well as the deoxy forms of these and othernucleotides.

[0091] The following examples are offered solely for the purposes ofillustration, and are intended neither to limit nor to define theinvention.

EXAMPLES

[0092] Examples 1-5 demonstrate the synthesis of sialyl lactose and itspurification using nanofiltration and ion exchange. In summary,N-acetyl-D-mannosamine (ManNAc) was generated fromN-acetyl-D-glucosamine (GluNAc) under basic conditions. The ManNAc wascondensed with sodium pyruvate to produce sialic acid enzymatically. Thesialyltransferase cycle was used to convert the sialic acid into sialyllactose, which was then purified by nanofilfration and ionic exchange.Example 6 demonstrates the separation of organics and inorganic salts bynanofiltration. Example 7 demonstrates the separation characteristics ofpolybenzamide nanofiltration membranes. Example 8 demonstrates theseparation characteristics of polyamide nanofiltration membranes.

Example 1 Synthesis and Purification of Sialic Acid

[0093] This example demonstrates a method for synthesizing sialic acidusing a relatively inexpensive substrate, GlcNAc, rather than the moreexpensive ManNAc or sialic acid. A procedure similar to that describedin Simon et al., J. Am. Chem. Soc. 110:7159 (1988), was used to convertGlcNAc to ManNAc. Briefly, GlcNAc (1000 g, 4.52 mole) was dissolved inwater (500 ml). The pH was adjusted to 12.0 with 50% NaOH (˜115 ml). Thesolution was stirred under argon for 7.5 hours, then cooled in an icebath and the pH was adjusted to 7.7 with concentrated HCl (˜200 ml).Sialic acid was then produced by aldol condensation of ManNAc.

[0094] To obtain sialic acid, the ManNAc produced in the previous stepwas subjected to aldol condensation mediated by N-acetylneuraminic acid(Neu5Ac) aldolase and pyruvic acid. To a 1.5 L aqueous solutioncontaining approximately 57g (0.258 mol) ManNAc and 193 g GlcNAc frombase-catalyzed epimerization was added 123.8 g sodium pyruvate (1.125mole), 1.5 g bovine serum albumin, and 0.75 g sodium azide. The pH wasadjusted to 7.5 and 11,930 U of sialic acid aldolase was added. Thesolution was incubated at 37° C. for 7 days. HPLC analysis on an AminexHPX87H (BioRad) column (0.004 M H₂SO₄, 0.8 ml/min, monitor A₂₂₀)revealed that the solution contained 0.157 M sialic acid (91% conversionof ManNAc, 0.235 mol).

Example 2 Synthesis of Sialyl Lactose Using Sialyltransferase Cycle

[0095] To the sialic acid produced in Example 1 was added lactosemonohydrate (79.2 g, 0.22 mol), 0.7 g bovine serum albumin,phosphoenolpyruvate monopotassium salt (37 g, 0.22 mol), and the pH wasadjusted to 7.5. CMP (2.84 g, 0.0088 mol), ATP (0.54 g, 0.0009 mol) wereadded, and the pH readjusted to 7.5. Sodium azide (0.35 g) was added, aswere the following enzymes: pyruvate kinase (19,800 U), myokinase(13,200 U), CMP sialic acid synthetase (440U, and sialyltransferase(165U). 66 ml of 1M MnCl₂ was added and the final volume adjusted to 2.2L with water. The reaction was carried out at room temperature.

[0096] The reaction was monitored daily by thin layer chromatography(TLC) and [Mn²⁺] was determined by ion chromatography.Additions/adjustments were made as shown in Table 1: TABLE 1 Day 2 44 ml1 M MnCl₂ added Day 4 43 ml 1 M MnCl₂ added Day 6 added 34.3 ml 1 MMnCl₂ , 37 g PEP; pH readjusted to 7.5; pyruvate kinase (19,800 U),myokinase (13,200 U), CMP sialic acid synthetase (440 U), andsialyltransferase (165 U) Day 7 31.7 ml 1 M MnCl₂ Day 8 24.6 ml 1 MMnCl₂ Day 9 44 ml 1 M MnCl₂ Day 10 30.8 ml 1 M MnCl₂ Day 11 31.7 ml 1 MMnCl₂ Day 12 24.6 ml 1 M MnCl₂, pH readjusted to 7.5 Day 13 440 U CMPsialic acid synthetase, 82.5 U sialyltransferase Day 14 pH readjusted to7.5 Day 16 37.7 ml 1 M MnCl₂, 19,800 U pyruvate kinase, 13,200 Umyokinase Day 17 26 g phosphenolpyruvate, trisodium salt

[0097] The sialyl lactose yield was approximately 70-80% as determinedby TLC.

Example 3 Synthesis of Sialyl Lactose Using Sialyltransferase Cycle

[0098] This example illustrates the production of α-N-acetylneuraminicacid(2,3)β-galactosyl(1,4)glucose using the sialyl transferase cyclewith control of the manganese ion concentration.

[0099] In a polypropylene vessel, phosphoenolpyruvate trisodium salt(285.4 g, 1.22 mol) and sialic acid (197 g, 0.637 mol) were dissolved in5 L of water and the pH was adjusted to 7.1 with 6 M NaOH.Cytidine-5′-monophosphate (5.14 g, 15.9 mmol) and potassium chloride(7.9 g, 0.106 mol) were added and the pH was re-adjusted to 7.45 with 6M NaOH. Pyruvate kinase (28,000 units), myokinase (17,000 units),adenosine triphosphate (0.98 g, 1.6 mmol), CMP NeuAc synthetase (1325units), α2,3 sialyltransferase (663 units) and MnCl₂.4H₂O (52.4 g, 0.265mol) were added and mixed. To a 3.7 L portion of the resulting mixturewas added lactose (119 g, 0.348 mol) and sodium azide (1.75 g). Thereaction mixture was kept at room temperature and monitored daily bythin layer chromatography (tlc) and ion chromatography. After two days,additional enzymes were added as follows: pyruvate kinase (38,100units), myokinase (23,700 units), CMP NeuAc synthetase (935 units), andα2,3 sialyltransferase (463 units). The pH was periodically adjusted to7.5 with 6 M NaOH. Additionally, the manganese ion concentration wasmeasured and supplemented as shown in Table 2 below. TABLE 2 AmountSupplemented [Mn⁺⁺] Loss of Mn⁺⁺ (mL of 1 M, Day (measured, mM) (fromprevious day) final added conc) 1 28 22.0 none 2 23.9 4.1 none 3 10.713.2 111 mL, +30 mM 4 1.4 39.3 111 mL, +30 mM 5 3.0 28.4 148 mL, +40 mM6 12.9 30.1  74 mL, +20 mM 7 10.0 22.9  80 mL, +20 mM 8 12.0 18.0  80mL, +20 mM 9 24.3 7.7 none

[0100] On day 9, the reaction was essentially complete by tlc. As theresults in the table indicate, the depletion of Mn⁺⁺ resulted inadditional amounts of MnCl₂.4H₂O being added almost daily to maintainthe metal ion concentration. Manganese ion is a required cofactor for atleast one enzyme in the sialyl transferase cycle. However, the manganeseion and the inorganic phosphate produced form a complex of very lowsolubility. Because of this limited solubility, the transferase cyclecan continue to proceed, but at reduced reaction rates. By supplementingthe manganese ions which are lost by precipitation with pyrophosphate,the rate of reaction can be maintained. Thus, when manganese ionconcentration is maintained in an optimal range, the sialyl transferasereaction cycle can be driven to completion.

Example 4 Purification of Sialyllactose Using Ion Exchange and ReverseOsmosis

[0101] This example illustrates the workup and purification of thetrisaccharide produced in Example 2 followed by peracetylation andesterification. A solution (2L) of sodium5-acetamido-3,5-dideoxy-α-D-glycero-D-galacto-nonulopyranosylonate-(2-3)-O-β-D-galactopyranosyl-(1-4)-O-β-D-glucopyranoseproduced from the action of a sialyl transferase in the presence of theappropriate cofactors on lactose (55 g) was filtered through paper. Thefiltrate was run through a membrane with a 3000 or 10,000 molecularweight cut off to remove protein from the desired product. The eluatewas concentrated and desalted by running it against a polyamide reverseosmosis membrane in a suitable apparatus (Cat. No. CDRN500 60,Millipore, Bedford, Mass.). The retentate containing the product wasevaporated to a thick syrup. Optionally the retentate can be treatedwith a chelating resin to remove divalent cations. After filtration thefiltrate contained the desired product substantially free of salts andin a high state of purity as shown by ¹Hmr spectroscopy. Otherwise thesyrup was so evaporated twice with pyridine (2×200 mL). The evaporationflask was charged with a solution of N,N-dimethylaminopyridine (2.2 g)in pyridine (1.2 L). Acetic anhydride (0.83 L) was added during a periodof 1 hour. The resulting mixture was left for 24-48 hours rotatingslowly at room temperature. The reaction is checked by TLC(methanol:dichloromethane 1:9). Upon complete reaction vacuum is appliedand the solution is evaporated to give a residue.

[0102] The residue was dissolved in ethyl acetate (1.5 L). This solutionwas washed with 5% aqueous hydrochloric acid (1.5 L) followed bysaturated aqueous sodium bicarbonate (1.5 L) and finally water (1.5 L).The organic layer was dried over anhydrous sodium sulfate and filtered.The filtrate was concentrated to a semi-solid residue. Theper-O-acetylated lactone trisaccharide (69 g) was dissolved in methanol(350 mL) and a sodium methoxide solution (17.5 mL, 25% solution inmethanol) was added followed by water (3.5 mL). When TLC developed withisopropanol:ammonium hydroxide:water 7:1:2 showed the reaction to becomplete acetic acid (2 mL) was added to the solution. Ethyl ether (180mL) was added to the solution to precipitate the product. This solid wasfiltered and dissolved in water (350 mL). Charcoal (24 g) was added tothis solution and heated to 60° C. for one hour. This solution wasallowed to cool to ambient temperature and filtered. Evaporation of thefiltrate gave the solid product (34 g). ¹H-NMR spectroscopy showed thissolid to be pure sialyl lactose containing 11% sodium acetate weight byweight.

Example 5 Purification of Sialyl Lactose Using Nanofiltration

[0103] A reaction mixture similar to that described in Example 2 wassubjected to filtration using an ultrafiltration membrane having a MWCOof 10 kDa to remove the proteins. The phosphate concentration [PO₄ ³⁻],as determined by a standard phosphorus assay procedure described below,was greater than 2.8 mM.

[0104] The solution was adjusted with concentrated HCl (˜500 ml) to pH=3.0. It was then purified on the Osmonics 213T membrane purificationmachine (membrane type MX07) at pH=3 for 5 hours until the conductivityof the permeate solution remained unchanged. The solution was thenrinsed from the machine and the combined rinse and feed solution treatedwith NaOH until pH 7.4. The Mn²⁺ concentration was measured by HPLC, asdescribed below. The nanofiltration parameters were as follows:Operation pressure: P_(f) = 100 psi Concentrate Flow Rate: Q_(c) = 5 GPMPermeate Flow Rate: Q_(f) = 7 GPH Temperature range: 20-40° C. Volume: 5Gallons

[0105] The conductivity of the initial permeate was 28.1 mS; after 5hours of recirculation, the conductivity had dropped to 115 μS, thephosphate concentration [PO₄ ³⁻] had decreased to 770 μM, and themanganese concentration [Mn²⁺] was 3.4 mM.

[0106] The solution was then adjusted to pH 7.4 and further purified onthe membrane purification machine (Osmonics, membrane type MX07) forabout 1 hour until the conductivity of the permeate solution remainedunchanged. The solution was then rinsed out from the membrane machine.The nanofiltration parameters were: Operation pressure: P_(f) = 100 psiConcentrate Flow Rate: Q_(c) = 5 GPM Permeate Flow Rate: Q_(f) = 0.3 GPMTemperature range: 20-40° C. Volume: 5 Gallon

[0107] The results of the filtration were as follows:

[0108] Conductivity: initial permeate conductivity: 2.01 mS after 5hours recirculation: 93.7 μS

[0109] Phosphate Concentration: [PO₄ ³⁻]=410 μM

[0110] Manganese Concentration: [Mn²⁺]=3.0 mM

[0111] The above solution (6 Gal) was then treated with AG50WX8 (H⁺)resin (BioRad, 1.18 Kg) and stirred for 2 hours until pH=2.0. The resinwas then filtered to provide a very light yellow solution. Only minimalamount of [Mn²⁺] was detected by HPLC. The solution was then neutralizedwith NaOH (50% w/w) to a pH of 7.4.

[0112] Before resin treatment: [Mn²⁺]=3 mM; [PO₄ ³⁻]=410 μM

[0113] After resin treatment:

[0114] pH=3, [Mn²⁺]=1.23 mM;

[0115] pH=2, [Mn²⁺]=6.8 μM; [PO₄ ³⁻]=190 μM

[0116] Some small portions of the above solution were treated with AG1X8(acetate form) resin to further remove the phosphate. The results areshown in Table 3 below: TABLE 3 Sample Volume (ml) Weight of resin (g)Stirring Time (hour) [PO₄ ³⁻] μM) 50 ml 0.25 g 1 86 50 ml 0.5 g 1 41 50ml 1.0 g 1 30 50 ml 2.0 g 1 8

[0117] The solution was further purified by recirculation of thesolution using an Osmonic membrane purification machine (Osmonic MX07)for 5 hours under the following conditions:

[0118] Operation pressure: P_(f)=100 psi

[0119] Concentrate Flow Rate: Q_(c)=5 GPM

[0120] Permeate Flow Rate: Q_(f)=0.2 GPM

[0121] Temperature range: 20- 40° C.

[0122] Volume: 5 Gallon

[0123] Results were as follows:

[0124] Permeate Conductivity: initial permeate conductivity: 0.136 mSafter 5 hours' separation: 45 μS

[0125] The solution was then concentrated to 3-4 L, after whichactivated charcoal (J. T. Baker, 180 g) was added. The suspension washeated at 55° C. for 2 hours. Charcoal was then removed by filtration toyield a very light yellow solution, which was lyophilized to a whitesolid.

[0126] Analysis data for the sialyl lactose solution purified asdescribed above are shown in Table 4. TABLE 4 Assay Result Method PO₄ ³⁻content 330 ppm (by weight) Phosphate assay¹ Nucleotide/ a)Not detected(ABS₂₈₀ = 0.0) UV (0.1 mM, nucleoside content b)Not detectedsialylactose) ¹H-NMR Mn²⁺ content 80 ppm (by weight) Determined by HPLC²Sialyl lactose 71% ¹H-NMR (1,2- content isopropylidene D-glucosefuranose was used as a standard Sialic acid content ˜2% ¹H-NMR Lactosecontent Not detectable ¹H-NMR Acetate content Not detectable ¹H-NMRN-acetyl Not detectable ¹H-NMR glucosamine content Pyruvate content Notdetectable ¹H-NMR

[0127] The unknown sample (100 μl) was diluted with D.I. water (775 μl).The solution was then treated with 100 μl of acid molybdate (prepared bydissolving 1.25 g of ammonium molybdate in 100 μl of 2.5N H₂SO₄), 25 μlof Fiska Subha Row Solution (purchased from Sigma as a powder, andprepared according to manufacturer's directions). The mixture was heatedat 100° C. for 7 min, the absorption at 810 nm was then recorded. Theconcentration was determined by comparing the absorption with aphosphate standard curve.

[0128]²HPLC Assay for the determination of Mn²⁺ concentration:

[0129] Column: Alitech Universal Cation column, 0.46×10 cm

[0130] Detector: Alltech model 320 conductivity detector

[0131] Mobile phase: 3mM phthalic acid, 0.5 mM dipicolinic acid

[0132] Flow rate: 1.5 ml/min

[0133] Column oven temperature: 35° C.

Example 6 Separation of Organics and Inorganic Salts by Nanofiltration

[0134] Various nanofiltration membranes were tested for ability toseparate various organic compounds and inorganic salts from an aqueoussolution. The membranes were tested at two different pHs to demonstratethat by adjusting the ionic charge of certain compounds, the separationprofile can be modulated. Results are shown in Table 5.

[0135] The nanofiltration membranes tested were the MX07, SX12, and B006produced by Osmonics, Inc. (Minnetonka Minn.) and the DL2540 produced byOsmonics, DeSalination Systems. The MX07 membrane was used as describedin Example 5 above. Parameters for the remaining membranes were as shownin Table 6. TABLE 5 Percentage of Compound Passing Through Membrane in30 Minutes Membrane MX07^(a) SX12^(a) B006^(a) DL2540^(a) Compound pH7.5 pH 3.0 pH 7.5 pH 3.0 pH 7.5 pH 3.0 pH 7.5 pH 3.0 Sodium 10 46 20 3915 64 1.8^(b) Phosphate Manganese 86 40 40 92 92 Sodium 35 59 45 65 3465 Pyruvate GlcNAc 70 28 84 12 Lactose 36 <5 pass Raffinose 0 0 8 52Sialic Acid 12 5 <1 1 Sodium 56 CMP <1 <1 PEP <1 8

[0136] TABLE 6 SX12 B006 DL2540 Pressure (P_(f)) (PSI) 200 100 200Concentrate Flow Rate (Q_(c)) 4.5 4 4 (GPM) Permeate Flow Rate (Q_(f))0.2 0.5 0.6 (GPM) Temperature Range (° C.) 20-40 20-40 20-40 Volume(Gal) 5 5 5

Example 7 Separation Characteristics of Polybenzamide NanofiltrationMembranes

[0137] This Example describes experiments which demonstrate that apolybenzamide membrane (YK, Osmonics) is effective for the purificationof sugars, in both flat-sheet and spiral-wound forms. The membrane wastested at varying pH levels for the passage or retention of sugars andsalts.

[0138] Materials and Methods

[0139] A. Flat Sheet and Spiral Wound Machine Operations and MembranePreparation

[0140] A Desal membrane machine (Osmonics, Desalination Systems,Escondido, Calif.) with membrane YK was washed thoroughly by firstrinsing the machine 4 to 5 times, each with approximately 1 L ofdistilled water. The water was poured into the feed tank, circulated forabout a minute (˜100 psi), and emptied using the drain valve, twistingit counterclockwise to an open position. The valve was closed afteremptying, and the process was repeated 4 to 5 times. After rinsing,approximately 1 more L of water was added. The system was recirculatedat a pressure of 150 psi for 30 min and then was emptied. The systemincluding the membrane was then used in the following experiments.

[0141] After the completion of each experiment, the machine was washedwith water 3 to 4 times as described above. Then, about IL of water wasrecirculated for about 15-20 minutes at 100-150 psi and emptied from themachine. Occasionally this was followed by an extra brief washing, ifsome of the test compound was suspected to still remain in theapparatus. The conductivity was always checked to make sure that all thesample was removed. If the conductivity remained high, the machine waswashed until the contaminants were virtually undetectable. Most of theionic compounds were removed easily, with the exception of MnCl₂, whichonly required 1 or 2 extra short washings. 5 B. Testing of Salts

[0142] To determine the retention characteristics of various salts, 10mM solutions of the following salts were tested with the flat sheetmembranes: MnCl₂, NaH₂PO₄, NaC₃H₃O₃, NaOAc, Na₄P₂O₇, sodium benzoate,MgSO₄, NaN₃, and NaCl. A 1L solution of one of the salts was poured intothe feed tank and recirculated at 100 psi for about 15 min. At thispoint, samples of both the permeate and the concentrate were collectedand measured using a conductivity meter. The samples were collectedevery five minutes thereafter, with a total of at least threecollections for each sample run. The percentage of salt passing throughthe membrane (the “percentage pass”) was calculated by dividing theconductivity of the permeate by the conductivity of the concentrate.

[0143] After the first run was completed, the pH of the solution wasthen lowered to pH 3.0, when possible, using a conjugate acid of thesalt being tested. The solution was recirculated while adjusting the pHto assure that the solution inside the machine was mixed as well. Thetesting process was repeated, with conductivity of both the permeate andthe concentrate being determined. The solution was then brought to a pHof about 7.0 with a conjugate base, and once again the run was repeatedat the new pH. Again, conductivity of both the permeate and concentratewas determined.

[0144] C. Testing of Sugars

[0145] Sugars that were tested included sialyl lactose, lactose,N-acetyl glucosamine, NeuAcα2,3Galβ1,4(Fucα1,3)GlcNAcβ1,4Galβ1-OEt(Compound I), Galα1,3Galβ1,4GlcNAcβ-O—(CH₂)₅—COOH (Compound II), LNT-2,LNnT, CMP, cytidine, and sialic acid. A sugar solution (1 L) was pouredinto the feed container and recirculated at 100 psi for at least 10minutes. Samples of the permeate and concentrate were taken at 10 min,and another sample of the permeate was taken at 15 min. The samples werecompared visually by TLC. Any pH adjustments that were made were byusing HCl and/or NaOH.

[0146] Results:

[0147] A. Flat Sheet Membrane

[0148] The retention characteristics for various salts and sugars of aflat sheet polybenzamide nanofiltration membrane (YK 002 on YV+ paperbacking (Osmonics) are shown in Table 7. The experiments were conductedat a temperature of 25-35° C. and a permeate flow rate of 2-8 mL/min.TABLE 7 Pressure % Pass* Material Concentration (psi) pH 3.0 pH 5** pH 7MnCl₂ 10 mM 100 66 12 9.8 NaH₂PO₄ 10 mM 100 82 15 4.6 NaPyruvate 10 mM100 80 36 9.8 NaCl 10 mM 100 — — 18 Sialyl lactose***  10 g/L 100 0 — 0Compound I***  10 g/L 100 0 — 0 Compound II***   2 g/L 100 0 — 0LNT-2***  .4 g/L 100 0 — 0 LNnT*** .35 g/L 100 0 — 0 Lactose  10 g/L 1000.0 0.3 — GlcNAc  10 g/L 100 5.9 — 3.7 Na₄P₂O₇ 10 mM 100 19 2.0 1.4Sialic Acid*** 10 mM 100 0 — — Cytidine***   1 g/L 100 0 — trace CMP***  1 g/L 100 0 — 0 Benzyl Alcohol*** 1.5% vol 100 — — 100 NaN₃ 10 mM 10081 — 67 MgSO₄ 10 mM 100 38 — 2.9 Benzoic acid ˜0.5 g/L   100 99 — — NaBenzoate 2.5% 100 — — 42

[0149] B. Spiral Wound Membrane

[0150] The retention characteristics for various salts and sugars of aspiral wound polybenzamide nanofiltration membrane (YK1812CZA; Osmonics)are shown in Table 8. The experiments were conducted at a temperature of25-35° C. and a permeate flow rate of 3 mL/sec. TABLE 8 Con- Pressure %Pass* Material centration (psi) pH 3** pH 5** pH 7** MnCl₂ 10 mM 100 50— 40 (pH 6.2) NaH₂PO₄ 10 mM 100 67 49 19 NaOAc 10 mM 100 — 81 65NaPyruvate 10 mM 100 81 — 26 NaCl 10 mM 100 79 78 — Sialyl lactose*** 10 g/L 100 0 — 0 Compound I***  10 g/L 100 0 — 0 Compound II***   2 g/L100 0 — 0 LNT-2*** 0.4 g/L 100 0 — 0 Lactose  10 g/L 100 0.59 — 2.3GlcNAc  10 g/L 100 13 7.1 19 Na₄P₂O₇ 10 mM 100 65 — 5.2 Sialic Acid***10 mM 100 0 — 0 Cytidine***   1 g/L 100 ˜10 — ˜5-10 CMP***   1 g/L 100trace — trace Sodium Benzoate ˜0.5 g/L   100 93 — 97

[0151] These results indicate that the YK002 flat sheet membrane and theYK1812CZA spiral wound membrane retained sialyl lactose as well asCompounds I and II, LNT-2, and LNnT, while allowing ionic compounds topass, making this membrane type a good choice for purification of suchsaccharides.

Example 8 Separation Characteristics of Polyamide NanofiltrationMembranes

[0152] This Example describes the evaluation of several polyamidemembranes for use in the purification of sugars, in both flat-sheet andspiral-wound forms. The membranes were tested at varying pH levels forthe passage or retention of sugars and salts.

[0153] Materials and Methods

[0154] A. Flat sheet and Spiral Wound Machine Operations and MembranePreparation:

[0155] A Desal membrane machine (Osmonics, Desalination Systems) with apolyamide membrane G-5 (GE; Osmonics) was washed thoroughly by firstrinsing the machine 4 to 5 times, each with approximately 1L ofdistilled water. The water was poured into the feed tank, circulated forabout a minute (˜100 psi), and emptied using the drain valve. The valvewas closed after emptying, and the process was repeated 4 to 5 times.After rinsing, approximately one more L of water was added. The systemwas recirculated at a pressure of 150 psi for 30 min and then wasemptied. The system including the membrane was then ready forapplication testing.

[0156] After each experiment, the machine was washed with water 3 to 4times as described above. Then, about 1 L of water was recirculated forabout 15-20 minutes at 100-150 psi and the machine was emptied.Occasionally this was followed by an extra brief washing, if some of thecompound was suspected to still remain in the apparatus. Theconductivity was always checked to make sure that all the sample wasremoved. If the conductivity remained high, the machine was washed untilthe contaminants were virtually undetectable. Most of the ioniccompounds were removed easily, with the exception of MnCl₂, which onlyrequired 1 or 2 extra short washings.

[0157] B. Testing of Salts

[0158] A 10 mM solution of the following salts were tested with the flatsheet membranes: MnCl₂, NaH₂PO₄, NaC₃H₃O₃, and NaCl. A 1L solution ofone of the salts was poured into the feed tank and recirculated at 100psi for about 15 min. At this point, samples of both the permeate andthe concentrate were collected and measured using a conductivity meter.The samples were collected every five minutes thereafter, with a totalof at least three collections for each sample run. The percentage passwas calculated by dividing the conductivity of the permeate by theconductivity of the concentrate. After the run was completed, the pH ofthe solution was lowered to pH 3.0, when possible, using a conjugateacid of the salt being tested. The solution was recirculated whileadjusting the pH to assure that the solution inside the machine wasmixed as well. The testing process was repeated, collecting data asbefore. Then the solution was brought to a pH of about 7.0 with aconjugate base, and once again the run was repeated at the new pH. Themachine was then emptied and rinsed as described above.

[0159] C. Testing of Sugars

[0160] The sugars that were tested included sialyl lactose, lactose,NeuAcα2,3Galβ1,4(Fucα1,3)GlcNAcβ1,4Galβ1-OEt (Compound I),Galα1,3Galβ1,4GlcNAcβ-O—(CH₂)₅—COOH (Compound II), LNT-2, and LNnT. Asugar solution (1L) was poured into the feed container and recirculatedat 100 psi for at least 10 minutes. Samples of the permeate andconcentrate were taken at 10 min, and another sample of the permeate wastaken at 15 min. The samples were compared visually by TLC. Any pHadjustments that were made were by using HCl and/or NaOH. After thesugar had been tested, it was transferred into a Pyrex flask to bereused for other membranes.

[0161] Results

[0162] A. Flat Sheet Membrane

[0163] The retention characteristics for various salts and sugars of aflat sheet polyamide nanofiltration membrane (G-10 (GH; Osmonics) areshown in Table 9. The A-value of the membrane was 10.0, and the percenttransmission of tap water was 62.8 (tested using 2000 ppm MgSO₄ atambient temperature). The experiments were conducted at a temperature of25-35° C. and a permeate flow rate of 5-8 mL/min. TABLE 9 Pressure %Pass* Material Concentration (psi) pH 3 pH 5** pH 7 MnCl₂ 10 mM 200 82.482.4 84.6 NaH₂PO₄ 10 mM 200 33.0 18.0 10.5 NaPyruvate 10 mM 200 49.4 —8.9 NaCl 10 mM 200 — — 17.8 Sialyl lactose***   10 g/L 200 <5 — <5Compound I***   10 g/L 200 — — 0 Compound II***   2 g/L 200 0 — —LNT-2***  0.4 g/L 200 — — trace^(#) LNnT*** 0.35 g/L 200 — — trace^(#)Lactose   10 g/L 200 2.0 — 4.2

[0164] In another experiment, a G-10 (GH) polyamide membrane with anA-value of 8.0 and a percent transmission of tap water of 38.9 wastested. The experiment was conducted at 25-35° C. and a permeate flowrate of 6-8 mL/min. The results are shown in Table 10. TABLE 10 Pressure% Pass* Material Concentration (psi) pH 3 pH 5** pH 7 MnCl₂ 10 mM 20070.8 — 77.7 NaH₂PO₄ 10 mM 200 39.4 32.1 16.2 NaPyruvate 10 mM 200 60.8 —21.8 NaCl 10 mM 200 — — 14.2 Sialyl lactose***   10 g/L 200 trace^(#) —trace^(#) Compound I***   10 g/L 200 — — 0 Compound II***   2 g/L 200trace^(#) — — LNT-2***  .4 g/L 200 — — trace^(#) LNnT*** 0.35 g/L 200 —— trace^(#) Lactose   10 g/L 200  3.8 — 22.1

[0165] A G-5 (GE) polyamide membrane (A-value: 3.9, percent transmissionof tap water: 33.9) was also tested. The experiment was conducted at25-35° C. and a permeate flow rate of 3-5 mL/min. Results are shown inTable 11. TABLE 11 Pressure % Pass* Material Concentration (psi) pH 3 pH5** pH 7 MnCl₂ 10 mM 200 77.6 80.1 81.8 NaH₂PO₄ 10 mM 200 30.0  8.6  4.8NaPyruvate 10 mM 200 48.2 —  8.4 NaCl 10 mM 200 — — 15.0 Sialyllactose***   10 g/L 200 0 — 0 Compound I***   10 g/L 200 — — 0 CompoundII***   2 g/L 200 0 — — LNT-2***  0.4 g/L 200 — — 0 LNnT*** 0.35 g/L 200. — — 0 Lactose   10 g/L 200 6.3 — 15.1

[0166] The sugar and salt retention characteristics of an HL (Osmonics)polyamide membrane are shown in Table 12. The experiments were carriedout at 25-35° C. and a permeate flow rate of 8-13 mL/min. TABLE 12Pressure % Pass* Material Concentration (psi) pH 3 pH 5** pH 7 MnCl₂ 10mM 100 48 22 23 NaH₂PO₄ 10 mM 100 67 24 7.5 NaPyruvate 10 mM 100 76 2916 NaCl 10 mM 100 71 66 — Sialyl lactose*** 10 g/L 100 0 — 0 Lactose 10g/L 100 1.9 4.1 —

[0167] B. Spiral Wound Membrane

[0168] The characteristics of sugar and salt retention on several spiralwound polyamide membranes were also determined. A GH1812CZA membrane(Osmonics) was tested at a temperature of 25-35° C. and a permeate flowrate of 1.5-2 mL/sec. Results are shown in Table 13. TABLE 13 Pressure %Pass* Material Concentration (psi) pH 3 pH 5** pH 7 MnCl₂ 10 mM 100 9394 — NaH₂PO₄ 10 mM 100 69 29 19 NaPyruvate 10 mM 100 68 — 42 NaCl 10 mM100 66 61 64 Sialyl lactose***  10 g/L 100 trace^(#) — trace^(#)Compound I***  10 g/L 100 0 — 0 Compound II***   2 g/L 100 0 — 0LNT-2*** 0.4 g/L 100 trace^(#) — trace^(#) Lactose  10 g/L 100 73 — 34GlcNAc  10 g/L 100 48 — 56 Na₄P₂O₇ 10 mM 100 13 — 5.7 Sialic Acid*** 10mM 100 25-50 — — Cytidine***   1 g/L 100 >50 — >50 CMP***   1 g/L100 >50 — >50 Benzoic Acid ˜0.5 g/L   100 90 — —

[0169] Results obtained for a GE1812CZA spiral wound polyamide membrane(Osmonics) tested at 25-35° C. and a decreased permeate flow rate of 0.9mL/sec are shown in Table 14. TABLE 14 Pressure % Pass* MaterialConcentration (psi) pH 3 pH 5** pH 7 MnCl₂ 10 mM 100 90 94 — NaH₂PO₄ 10mM 100 54 14 8.7 NaOAc 10 mM 100 98 — 24 NaPyruvate 10 mM 100 73 — 45NaCl 10 mM 100 54 — 44 Sialyl lactose***  10 g/L 100 0 — 0 Compound I*** 10 g/L 100 0 — 0 Compound II***   2 g/L 100 0 — 0 Lactose  10 g/L 10041 — 43 GlcNAc  10 g/L 100 72 — 69 MgSO₄ 10 mM 100 50 37 — Na₄P₂O₇ 10 mM100 11 — 4.7 Sialic Acid*** 10 mM 100 trace^(#) — trace^(#) Cytidine***  1 g/L 100 >50 — >50 CMP***   1 g/L 100 >50 — >50 Benzoic Acid ˜0.5g/L   100 63 40 —

[0170] These results demonstrate that the G-10 (GH) (A value=10) and theG-10 (GH) (A value=8) flat sheet membranes and the GH1812CZA spiralwound membrane allowed ions to pass but did not efficiently retainsialyl lactose or similar trisaccharides. The G-5 (GE) (A-value=3.9)flat sheet membrane and the GE1812CZA spiral wound membrane retainedsialyl lactose as well as Compounds I and II, LNT-2, and LNnT, whileallowing ionic compounds to pass.

[0171] All publications, patents and patent applications mentioned inthis specification are herein incorporated by reference into thespecification to the same extent as if each individual publication,patent or patent application was specifically and individually indicatedto be incorporated herein by reference.

[0172] The above description is illustrative and not restrictive. Manyvariations of the invention will become apparent to those of skill inthe art upon review of this disclosure. Merely by way of example anumber of substrates, enzymes, and reaction conditions can besubstituted into the glycosyl transferase cycles as part of the presentinvention without departing from the scope of the invention. The scopeof the invention should, therefore, be determined not with reference tothe above description, but instead should be determined with referenceto the appended claims along with their full scope of equivalents.

What is claimed is:
 1. A method of purifying a carbohydrate compound from a feed solution containing a contaminant, the method comprising contacting the feed solution with a nanofiltration or reverse osmosis membrane under conditions such that the membrane retains the carbohydrate compound while a majority of the contaminant passes through the membrane, wherein the carbohydrate compound is selected from the group consisting of: sialyl lactosides; sialic acid; LnNT; LNT-2; NeuAcα(2→3)Galβ(1→4)(Fucα1→3)Glc(R¹)β1-OR², wherein R¹ is OH or NAc; R² is a hydrogen, an alkoxy, a saccharide, an oligosaccharide or an aglycon group having at least one carbon atom; and Galα(1→3)Galβ(1→4)Glc(R¹)β-O—R³, wherein wherein R¹ is OH or NAc; R³ is —(CH₂)_(n)-COX, with X═OH, OR⁴, —NHNH₂, R⁴ being a hydrogen, a saccharide, an oligosaccharide or an aglycon group having at least one carbon atom, and n=an integer from 2 to
 18. 2. The method of claim 1, wherein the carbohydrate compound is selected from the group consisting of a sialyl lactoside, LnNT, LNT-2, and Galα1,3Galβ1,4GlcNAcβ1-O—(CH₂)₅—COOH.
 3. The method of claim 2, wherein the carbohydrate compound is selected from the group consisting of a sialyl(α2,3)-lactoside and a sialyl(α2,6)-lactoside.
 4. The method according to claim 2, wherein the membrane comprises polyamide or polybenzamide.
 5. The method according to claim 4, wherein the membrane is a membrane selected from the group consisting of a YK, a GE (G-5), and an MX07.
 6. The method of claim 1, wherein the carbohydrate compound is NeuAcα2,3Galβ1,4(Fucα1,3)GlcNAcβ1,4Galβ1-OEt.
 7. The method according to claim 6, wherein the membrane is a membrane selected from the group consisting of a YK, a GE (G-5), a GH (G-10), an HL, and an MX07.
 8. The method of claim 1, wherein the feed solution is an enzymatic reaction mixture used for synthesis of the carbohydrate compound.
 9. The method of claim 1, wherein the ability of the membrane to retain the carbohydrate compound while passing the contaminant is optimized by adjusting the pH of the feed solution.
 10. A method of purifying a carbohydrate compound from a feed solution containing a contaminant, the method comprising contacting the feed solution with a nanofiltration or reverse osmosis membrane under conditions such that the membrane retains the carbohydrate compound while a majority of the contaminant passes through the membrane, wherein the carbohydrate compound has the formula

wherein Z is hydrogen, C₁-C₆ acyl or

Y is selected from the group consisting of C(O), SO₂, HNC(O), OC(O) and SC(O); R¹ is selected from the group consisting of an aryl, a substituted aryl and a phenyl C₁-C₃ alkylene group, wherein said aryl substitutent is selected from the group consisting of a halo, trifuloromethyl, nitro, C₁-C₁₈ alkyl, C₁-C₁₈ alkoxy, amino, mono-C₁-C₁₈ alkylamino, di-C₁-C₁₈ alkylamino, benzylamino, C₁-C₁₈ alkylbenzylamino, C₁-C₁₈ thioaklyl and C₁-C₁₈ alkyl carboxamido groups, or R¹Y is allyloxycarbonyl or chloroacetyl; R² is selected from the group consisting of monosaccharide (including β1,3Gal—OR, where R═H, alkyl, aryl or acyl), disaccharide, hydrogen, C₁-C₁₈ straight chain, branched chain or cyclic hydrocarbyl, C₁-C₆ alkyl, 3-(3,4,5-trimethoxyphenyl)propyl, C₁-C₅ alkylene ω-carboxylate, ω-trisubstituted silyl C₂-C₄ alkylene wherein said ω-trisubstituted silyl is a silyl group having three substituents independently selected from the group consisting of C₁-C₄ alkyl, phenyl, or OR² together form a C₁-C₁₈ straight chain, branched chain or cyclic hydrocarbyl carbamate; R³ is hydrogen or C₁-C₆ acyl; R⁴ is hydrogen, C₁-C₆ alkyl or benzyl; R⁵ is selected from the group consisting of hydrogen, benzyl, methoxybenzyl, dimethoxybenzyl and C₁-C₆ acyl; R⁷ is methyl or hydroxymethyl; and X is selected from the group consisting of C₁-C₆ acyloxy, C₂-C₆ hydroxylacyloxy, hydroxy, halo and azido.
 11. The method of claim 10, wherein the carbohydrate compound has a formula NeuAcα(2→3)Galβ(1→4)GlcN(R¹)β-OR² or NeuAcα(2→3)Galβ(1→4)GlcN(R¹)β(1→3)Galβ-OR², wherein R¹ is alkyl or acyl from 1-18 carbons, 5,6,7,8-tetrahydro-2-naphthamido; benzamido; 2-naphthamido; 4-aminobenzamido; or 4-nitrobenzamido, and R² is a hydrogen, a saccharide, an oligosaccharide or an aglycon group having at least one carbon atom.
 12. The method of claim 10, wherein the carbohydrate compound has a formula NeuAcα(2→3)Galβ(1→4)(Fucα1→3)GlcN(R¹)β-OR² or NeuAcα(2→3)Galβ(1→4) (Fucα1→3)GlcN(R¹)β(1→3)Galβ-OR², wherein R¹ is alkyl or acyl from 1-18 carbons, 5,6,7,8-tetrahydro-2-naphthamido; benzamido; 2-naphthamido; 4-aminobenzamido; or 4-nitrobenzamido, and R² is a hydrogen, a saccharide, an oligosaccharide or an aglycon group having at least one carbon atom.
 13. A method of purifying a carbohydrate compound from a feed solution comprising an enzymatic reaction mixture used to synthesize the carbohydrate compound, the method comprising the steps of: removing any proteins present in the feed solution by contacting the feed solution with an ultrafiltration membrane so that proteins are retained the membrane while the carbohydrate compound passes through the membrane as a permeate; and contacting the permeate from the ultrafiltration step with a nanofiltration or reverse osmosis membrane under conditions such that the nanofiltration or reverse osmosis membrane retains the carbohydrate compound while a majority of an undesired contaminant passes through the membrane.
 14. A method of purifying a carbohydrate compound from a feed solution containing a contaminant, the method comprising contacting the feed solution with a nanofiltration or reverse osmosis membrane under conditions such that the membrane retains the carbohydrate compound while a majority of the contaminant passes through the membrane, wherein the carbohydrate compound is a nucleotide or a nucleotide sugar.
 15. The method of claim 14, wherein the nucleotide sugar is selected from the group consisting of GDP-fucose, GDP-mannose, CMP-NeuAc, UDP-glucose, UDP-galactose, and UDP-N-acetylgalactosamine.
 16. The method of claim 14, wherein the carbohydrate compound is a nucleotide or nucleoside.
 17. A method for removal of a contaminant from a feed solution comprising a carbohydrate of interest, the method comprising contacting the feed solution with a first side of a nanofiltration or reverse osmosis membrane having rejection coefficients for the carbohydrate and the contaminant such as to retain the carbohydrate while allowing the contaminant to pass through the membrane, wherein the feed solution comprises an enzymatic reaction mixture used to synthesize the carbohydrate.
 18. The method according to claim 17, wherein the enzymatic reaction comprises enzymatic degradation of an oligosaccharide or polysaccharide.
 19. The method according to claim 17, wherein the membrane separates the solution into a retentate portion and a permeate portion, the retentate portion comprising a lower concentration of the contaminant relative to the contaminant concentration in the feed solution.
 20. The method according to claim 17, wherein the pH of the feed solution is selected so as to adjust rejection coefficients of the membrane for the carbohydrate compound and the contaminant to obtain increased removal of the contaminant from the feed solution and/or increased retention of the carbohydrate compound.
 21. The method according to claim 20, wherein the contaminant is an anion and the pH of the feed solution is between about 1 and about
 7. 22. The method according to claim 20, wherein the contaminant is a cation and the pH of the feed solution is between about 7 and
 14. 23. The method according to claim 20, wherein the pH of the feed solution is selected so as to make the net surface charge of the nanofiltration membrane positive.
 24. The method according to claim 20, wherein the pH of the feed solution is selected so as to make the net surface charge of the nanofiltration membrane negative.
 25. The method according to claim 20, wherein the pH of the feed solution is selected so as to make the net surface charge of the nanofiltration membrane neutral.
 26. The method according to claim 17, wherein the carbohydrate is an oligosaccharide or polysaccharide.
 27. The method according to claim 17, wherein the carbohydrate is sialylated.
 28. The method according to claim 17, wherein the contaminant comprises one or more compounds selected from the group consisting of nucleotide sugars, unreacted sugars, inorganic ions, pyruvate, phosphate, phosphoenolpyruvate, nucleotides, nucleosides, and proteins.
 29. The method according to claim 17, wherein proteins are removed from the mixture prior to nanofiltration.
 30. The method according to claim 17, which further comprises an ion exchange purification step. 